Enzyme granules for animal feed

ABSTRACT

The present invention is related to feed granules comprising a feed enzyme and a zinc salt of an organic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/834,343 filed Aug. 6, 2007, now pending, which claims priority or thebenefit under 35 U.S.C. 119 of Danish application no. PA 2006 01036filed Aug. 7, 2006 and U.S. provisional application No. 60/839,450 filedAug. 23, 2006. The content of these applications is fully incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to enzyme granules especially for animalfeed. The present invention further relates to the manufacturing of saidgranules.

BACKGROUND OF THE INVENTION

In the art concerning animal feed it is a well-known fact that pelletingof feed is a desideratum, as pelleting of feed increases thedigestibility of especially the starch fraction of the feed.Furthermore, it is known that pelleting of animal feed reduces dustproblems.

In the process of producing feed pellets it is considered necessary tosteam treat mash feed in order to kill pathogenic micro-organisms ifpresent and to partly gelatinize starch in order to improve the physicalproperties of the feed, whereby a steam treatment of around 70-120° C.is appropriate. Active compounds present in the feed such as enzymes arenot very stable at high temperatures and humidities, and thus, a largesurplus of enzymes has to be used, or enzyme free feed components arepelletized and steam treated, where after an enzyme containing slurry orsolution is coated onto the steam treated pellets. However, this coatingprocess is cumbersome and is often not compatible with feed millequipment.

An attempt to obtain improved enzyme granules for feed is found in WO92/12645. WO 92/12645 describes T-granules, which are coated with a fator a wax. Said T-granules are mixed with feed components steam treatedand subsequently pelletized. By this invention it was possible to heattreat the granules comprising enzymes and avoid the cumbersome coatingwith enzymes after the heat treatment. The use of wax coated T-granuleswas a significant improvement in this field as it was possible tomaintain an acceptable enzyme activity during steam pelleting. Anotherattempt to improve the pelleting stability of enzyme granules isdescribed in WO 2006/034710 where it has been found that coating enzymegranules with a salt coating improves the pelleting stability.

Some feed mills are run under very aggressive conditions which are veryharsh for the improved enzyme granules, thus there is still a demand forimproved pelleting stability.

The present invention provides a solution to this problem byincorporation of a zinc salt of an organic acid together with theenzyme.

It is known in the art to use inorganic salts of zinc and magnesium tostabilize enzyme granules for animal feed, see WO 97/05245. It hassurprisingly been found that the use of zinc salts of organic acidsincreases the enzyme stability both after granulation but also duringsteam pelleting.

SUMMARY OF THE INVENTION

One object of the present invention is to provide an enzyme granule withgood pelleting stability. A second object of the present invention is toprovide a simple process for obtaining enzyme granules with goodpelleting stability.

It has surprisingly been found that zinc salts of organic acids have avery positive effect on enzyme stability during pelleting, and per sestability if formulated together with the enzyme.

The present invention provides thus in a first aspect a granule suitablefor use in animal feed compositions comprising a feed enzyme and a zincsalt of an organic acid.

The present invention further provides feed compositions comprising theenzyme granule of the invention.

DETAILED DESCRIPTION OF THE INVENTION Introduction

We have surprisingly found it possible to increase the stability ofenzymes comprised in granules during steam pelleting by adding a zincsalt of an organic acid.

Definitions

By the term “solution” is meant a homogeneous mixture of two or moresubstances.

By the term “suspension” is meant fine particles suspended in a liquid.

By the term “particle size” is meant the mass mean diameter of thegranules.

By the term “enzyme core” is meant the raw granule comprising theenzyme, thus the granule before any coatings are applied.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. As used in the specificationand claims, the singular “a”, “an” and “the” include the pluralreferences unless the context clearly dictates otherwise. For example,the term granule may include a plurality of granules.

By “organic zinc salt” is meant a zinc salt of an organic acid.

The Granule

When referring to the granule of the present invention it can either bea single granule or several granules.

The granule of the present invention may have a matrix structure wherethe components are mixed homogeneously or it may be a layered granulecomprising a core and one or more layers surrounding the core.

The granule of the present invention which is particularly well suitedfor steam pelleting and as part of a steam treated pelletized feedcomposition, comprises an enzyme comprising region wherein a zinc saltof an organic acid is present to stabilize the enzyme. In a particularembodiment of the present invention the enzyme and the zinc salt of anorganic acid is present in a homogeneous matrix. The matrix comprisingthe enzyme and the zinc salt of an organic acid may comprise otherauxiliary components.

Suitable particle sizes of the granule of the present invention is foundto be 20-2000 μm, more particular 50-1000 μm. Even further 75-700 μm.The granule of the present invention may in a particular embodiment havea particle size below 700 μm. In another particular embodiment of thepresent invention the particle size of the finished granule is 100-800μm. In a more particular embodiment of the present invention theparticle size of the finished granule is 300-600 μm. In a mostparticular embodiment of the present invention the particle size is450-550 μm. In another particular embodiment of the present inventionthe particle size of the finished granule is below 400 μm. In anothermost particular embodiment the particle size of the granules of thepresent invention is above 250 μm and below 350 μm.

In a particular embodiment of the present invention the particle size ofthe granule of the present invention is between 210 and 390 μm.

The Enzyme Comprising Matrix

The enzyme comprising matrix or region of the granule may be the core ofthe granule, a layer surrounding the core, or the granule as such, ifthe structure of the granule is homogenous throughout the granule. Theremay be layers present between the enzyme layer and the core, or theenzyme layer may be next to the core. The core may be an inert particle.

The enzyme comprising region may be a homogeneous blend comprisingenzyme and a zinc salt of an organic acid. Said homogeneous blend mayeither constitute the core or it may constitute a matrix layersurrounding an inert core particle. The enzyme matrix may comprisefurther granulation agents.

The core particle, which either consists of a homogeneous blendcomprising the enzyme and the zinc salt of an organic acid, or consistof an inert core upon which a layer comprising the enzyme and zinc saltof an organic acid is applied has in a particular embodiment a particlesize of 20-800 μm. In a more particular embodiment of the presentinvention the core particle size is 50-500 μm. In an even moreparticular embodiment of the present invention the core particle size is100-300 μm. In a most particular embodiment of the present invention thecore particle size is 150-250 μm. In another particular embodiment ofthe present invention the core particle size is 400-500 μm.

The core particle comprising the enzyme has in a particular embodiment aparticle size of 20-800 μm. In a more particular embodiment of thepresent invention the core particle size is 50-500 μm. In an even moreparticular embodiment of the present invention the core particle size is100-300 μm. In a most particular embodiment of the present invention thecore particle size is 150-250 μm. In another particular embodiment ofthe present invention the core particle size is 400-500 μm.

Inert Core Particle:

Inert core particles such as placebo particles, carrier particles,inactive nuclei, inactive particles, non-pareil particles, non-activeparticles or seeds, are particles not comprising enzymes or only minoramount of enzymes upon which a coating mixture comprising the enzyme canbe layered. They may be formulated with organic or inorganic materialssuch as inorganic salts, sugars, sugar alcohols, small organic moleculessuch as organic acids or salts, starch, flour, treated flour, cellulose,polysaccharides, minerals such as clays or silicates or a combination oftwo or more of these.

In a particular embodiment of the present invention the particles to becoated are inactive particles. In a more particular embodiment of thepresent invention the material of the core particles is selected fromthe group consisting of inorganic salts, sugar alcohols, small organicmolecules, starch, flour, cellulose and minerals.

Inert particles can be produced by a variety of granulation techniquesincluding: crystallisation, precipitation, pan-coating, fluid bedcoating, fluid bed agglomeration, rotary atomization, extrusion,prilling, spheronization, size reduction methods, drum granulation,and/or high shear granulation.

Enzymes:

The feed enzyme in the context of the present invention may be anyenzyme or combination of different enzymes that are suitable to be givento an animal, meaning that it in one way or the other will be good forthe animal nutritionally to eat the enzyme. Accordingly, when referenceis made to “an enzyme” this will in general be understood to include onefeed enzyme or a combination of feed enzymes. In a particularembodiment, it is not construed as including enzymes which have atherapeutic function in medical sense.

The feed enzymes should be feed/food grade, thus meaning that they maynot be harmful to the animal and be a feed/food grade meaning that itshould comply with recommended purity specifications for food gradeenzymes. In a particular embodiment, this means that the enzyme compliesrecommended purity specifications for food grade enzymes given by theJoint FAO/WHO Expert Committee on Food Additives (JECFA) and the FoodChemical Codex (FCC).

The enzyme shall in a particular embodiment comprise less than 30coliform bacteria pr gram and comprise a viable count of less than50000/g.

The granules of the invention include between about 0.0005 to about 50%on a dry weight basis of the enzyme component of the granule. Forinstance, the weight percent of enzyme in embodiments of the inventioncomprises at least 0.0005 to about 25%, at least 0.001 to about 15%, atleast 0.01 to about 10%, at least 0.1 to about 10%, at least 1.0 toabout 10%, at least 1.0 to about 8%, at least 1.0 to about 5%, and atleast 2.0 to at least 5% in the granule. Typical doses of 25 to 400grams of the stable, enzyme granules per ton of feed will deliver about0.0001 to about 80 grams of active enzyme protein per ton of feed, andthe enzyme granules may be dosed as high as 5000 grams per ton of feed.

It is to be understood that enzyme variants (produced, for example, byrecombinant techniques) are included within the meaning of the term“enzyme”. Examples of such enzyme variants are disclosed, e.g., in EP251,446 (Genencor), WO 91/00345 (Novo Nordisk), EP 525,610 (Solvay) andWO 94/02618 (Gist-Brocades NV).

Enzymes can be classified on the basis of the handbook EnzymeNomenclature from NC-IUBMB, 1992), see also the ENZYME site at theinternet: www.expasy.ch/enzyme/. ENZYME is a repository of informationrelative to the nomenclature of enzymes. It is primarily based on therecommendations of the Nomenclature Committee of the International Unionof Biochemistry and Molecular Biology (IUB-MB), Academic Press, Inc.,1992, and it describes each type of characterized enzyme for which an EC(Enzyme Commission) number has been provided (Bairoch, 2000, The ENZYMEDatabase, Nucleic Acids Res. 28:304-305). This IUB-MB Enzymenomenclature is based on their substrate specificity and occasionally ontheir molecular mechanism; such a classification does not reflect thestructural features of these enzymes.

Another classification of certain glycoside hydrolase enzymes, such asendoglucanase, xylanase, galactanase, mannanase, dextranase andalpha-galactosidase, in families based on amino acid sequencesimilarities has been proposed a few years ago. They currently fall into90 different families: See the CAZy(ModO) internet site (Coutinho, P. M.& Henrissat, B. (1999) Carbohydrate-Active Enzymes server at URL:afmb.cnrs-mrs.fr/˜cazy/CAZY/index.html (corresponding papers: Coutinho,P. M. & Henrissat, B. (1999) Carbohydrate-active enzymes: an integrateddatabase approach. In “Recent Advances in Carbohydrate Bioengineering”,H. J. Gilbert, G. Davies, B. Henrissat and B. Svensson eds., The RoyalSociety of Chemistry, Cambridge, pp. 3-12; Coutinho, P. M. & Henrissat,B. (1999) The modular structure of cellulases and othercarbohydrate-active enzymes: an integrated database approach. In“Genetics, Biochemistry and Ecology of Cellulose Degradation”, K.Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita and T. Kimuraeds., Uni Publishers Co., Tokyo, pp. 15-23).

The types of enzymes which may be incorporated in granules of theinvention include oxidoreductases (EC 1.-.-.-), transferases (EC2.-.-.-), hydrolases (EC 3.-.-.-), lyases (EC 4.-.-.-), isomerases (EC5.-.-.-) and ligases (EC 6.-.-.-).

Preferred oxidoreductases in the context of the invention areperoxidases (EC 1.11.1), laccases (EC 1.10.3.2) and glucose oxidases (EC1.1.3.4). An example of a commercially available oxidoreductase (EC1.-.-.-) is Gluzyme™ (enzyme available from Novozymes A/S). Furtheroxidoreductases are available from other suppliers. Preferredtransferases are transferases in any of the following sub-classes:

a Transferases transferring one-carbon groups (EC 2.1);

b transferases transferring aldehyde or ketone residues (EC 2.2);acyltransferases (EC 2.3);

c glycosyltransferases (EC 2.4);

d transferases transferring alkyl or aryl groups, other that methylgroups (EC 2.5); and

e transferases transferring nitrogenous groups (EC 2.6).

A most preferred type of transferase in the context of the invention isa transglutaminase (protein-glutamine γ-glutamyltransferase; EC2.3.2.13).

Further examples of suitable transglutaminases are described in WO96/06931 (Novo Nordisk A/S).

Preferred hydrolases in the context of the invention are: carboxylicester hydrolases (EC 3.1.1.-) such as lipases (EC 3.1.1.3); phytases (EC3.1.3.-), e.g., 3-phytases (EC 3.1.3.8) and 6-phytases (EC 3.1.3.26);glycosidases (EC 3.2, which fall within a group denoted herein as“carbohydrases”), such as α-amylases (EC 3.2.1.1); peptidases (EC 3.4,also known as proteases); and other carbonyl hydrolases. Examples ofcommercially available phytases include Bio-Feed™ Phytase (Novozymes),Ronozyme™ product series (DSM Nutritional Products), Natuphos™ (BASF),Finase™ (AB Enzymes), and the Phyzyme™ product series (Danisco). Otherpreferred phytases include those described in WO 98/28408, WO 00/43503,and WO 03/066847.

In the present context, the term “carbohydrase” is used to denote notonly enzymes capable of breaking down carbohydrate chains (e.g.,starches or cellulose) of especially five- and six-membered ringstructures (i.e., glycosidases, EC 3.2), but also enzymes capable ofisomerizing carbohydrates, e.g., six-membered ring structures such asD-glucose to five-membered ring structures such as D-fructose.

Carbohydrases of relevance include the following (EC numbers inparentheses): α-amylases (EC 3.2.1.1), β-amylases (EC 3.2.1.2), glucan1,4-α-glucosidases (EC 3.2.1.3), endo-1,4-beta-glucanase (cellulases, EC3.2.1.4), endo-1,3(4)-β-glucanases (EC 3.2.1.6), endo-1,4-β-xylanases(EC 3.2.1.8), dextranases (EC 3.2.1.11), chitinases (EC 3.2.1.14),polygalacturonases (EC 3.2.1.15), lysozymes (EC 3.2.1.17),β-glucosidases (EC 3.2.1.21), α-galactosidases (EC 3.2.1.22),β-galactosidases (EC 3.2.1.23), amylo-1,6-glucosidases (EC 3.2.1.33),xylan 1,4-β-xylosidases (EC 3.2.1.37), glucan endo-1,3-β-D-glucosidases(EC 3.2.1.39), α-dextrin endo-1,6-α-glucosidases (EC3.2.1.41), sucroseα-glucosidases (EC 3.2.1.48), glucan endo-1,3-α-glucosidases (EC3.2.1.59), glucan 1,4-β-glucosidases (EC 3.2.1.74), glucanendo-1,6-β-glucosidases (EC 3.2.1.75), galactanases (EC 3.2.1.89),arabinan endo-1,5-α-L-arabinosidases (EC 3.2.1.99), lactases (EC3.2.1.108), chitosanases (EC 3.2.1.132) and xylose isomerases (EC5.3.1.5).

In the present context, a phytase is an enzyme which catalyzes thehydrolysis of phytate (myo-inositol hexakisphosphate) to (1)myo-inositol and/or (2) mono-, di-, tri-, tetra- and/or penta-phosphatesthereof and (3) inorganic phosphate.

Three different types of phytases are known: A so-called 3-phytase(alternative name 1-phytase; a myo-inositol hexaphosphate3-phosphohydrolase, EC 3.1.3.8), a so-called 4-phytase (alternative name6-phytase, name based on 1L-numbering system and not 1D-numbering, EC3.1.3.26), and a so-called 5-phytase (EC 3.1.3.72). For the purposes ofthe present invention, all three types are included in the definition ofphytase.

For the purposes of the present invention phytase activity may be,preferably is, determined in the unit of FYT, one FYT being the amountof enzyme that liberates 1 micro-mol inorganic ortho-phosphate per min.under the following conditions: pH 5.5; temperature 37° C.; substrate:sodium phytate (C₆H₆O₂₄P₆Na₁₂) in a concentration of 0.0050 mol/l.Suitable phytase assays are described in Example 1 of WO 00/20569. FTUis for determining phytase activity in feed and premix.

Preferred examples of phytases are microbial phytases, such as fungal orbacterial phytases, e.g., derived from the following:

i) Ascomycetes, such as those disclosed in EP 684313 or U.S. Pat. No.6,139,902; Aspergillus awamori PHYA (SWISSPROT P34753, Gene 133:55-62(1993)); Aspergillus niger (ficuum) PHYA (SWISSPROT P34752, Gene127:87-94 (1993), EP 420358); Aspergillus awamori PHYB (SWISSPROTP34755, Gene 133:55-62 (1993)); Aspergillus niger PHYB (SWISSPROTP34754, Biochem. Biophys. Res. Commun. 195:53-57 (1993)); Emericellanidulans PHYB (SWISSPROT 000093, Biochim. Biophys. Acta 1353:217-223(1997));

ii) Thermomyces or Humicola, such as the Thermomyces lanuginosus phytasedisclosed in WO 97/35017;

iii) Basidiomycetes, such as Peniophora (WO 98/28408 and WO 98/28409);

iv) Other fungal phytases such as those disclosed in JP 11000164(Penicillium phytase), or WO 98/13480 (Monascus anka phytase);

v) Bacillus, such as Bacillus subtilis PHYC (SWISSPROT 031097, Appl.Environ. Microbiol. 64:2079-2085 (1998)); Bacillus sp. PHYT (SWISSPROT066037, FEMS Microbiol. Lett. 162:185-191 (1998); Bacillus subtilis PHYT(SWISSPROT P42094, J. Bacteriol. 177:6263-6275 (1995)); the phytasedisclosed in AU 724094, or WO 97/33976;

vi) Escherichia coli (e.g., U.S. Pat. No. 6,110,719);

vii) Citrobacter, such as Citrobacter freundii (disclosed in WO2006/038062, WO 2006/038128, or with the sequence of UniProt Q676V7),Citrobacter braakii (disclosed in WO 2004/085638 (Geneseqp ADU50737),and WO 2006/037328), and Citrobacter amalonaticus or Citrobactergillenii (disclosed in WO 2006/037327);

viii) Other bacterial phytases such as the phytase from Buttiauxella(disclosed in WO 2006/043178);

ix) Yeast phytases, e.g., from Schwanniomyces occidentalis (e.g.,disclosed in U.S. Pat. No. 5,830,732); as well as

x) a phytase having an amino acid sequence of at least 75% identity to amature amino acid sequence of any one of the phytases of (i)-(ix);

xi) a variant of the phytase of (i)-(ix) comprising a substitution,deletion, and/or insertion of one or more amino acids;

xii) an allelic variant of the phytase of (i)-(ix);

xiii) a fragment of the phytase of (i)-(ix) that retains phytaseactivity; or

xiv) a synthetic polypeptide designed on the basis of (i)-(ix) andhaving phytase activity.

Preferred examples of phytase variants are disclosed in, e.g., WO99/49022, WO 99/48380, WO 00/43503, EP 0897010, EP 0897985, WO2003/66847, as well as in the above-mentioned WO 2006/038063, WO2006/038128, and WO 2006/43178).

Examples of commercially available proteases (peptidases) includeKannase™, Everlase™, Esperase™, Alcalase™, Neutrase™, Durazym™,Savinase™, Ovozyme™, Pyrase™, Pancreatic Trypsin NOVO (PTN), Bio-Feed™Pro and Clear-Lens™ Pro (all available from Novozymes A/S, Bagsvaerd,Denmark). Other preferred proteases include those described in WO01/58275 and WO 01/58276.

Other commercially available proteases include Ronozyme™ Pro, Maxatase™,Maxacal™, Maxapem™, Opticlean™, Propease™, Purafect™ and Purafect Ox™(available from Genencor International Inc., Gist-Brocades, BASF, or DSMNutritional Products).

Examples of commercially available lipases include Lipex™, Lipoprime™Lipopan™ Lipolase™, Lipolase™ Ultra, Lipozyme™ Palatase™, Resinase™,Novozym™ 435 and Lecitase™ (all available from Novozymes A/S).

Other commercially available lipases include Lumafast™ (Pseudomonasmendocina lipase from Genencor International Inc.); Lipomax™(Pseudomonas pseudoalcaligenes lipase from Gist-Brocades/Genencor Int.Inc.; and Bacillus sp. lipase from Solvay enzymes. Further lipases areavailable from other suppliers.

Examples of commercially available carbohydrases include Alpha-Gal™,Bio-Feed™ Alpha, Bio-Feed™ Beta, Bio-Feed™ Plus, Bio-Feed™ Wheat,Bio-Feed™ Z, Novozyme™ 188, Carezyme™, Celluclast™, Cellusoft™,Celluzyme™, Ceremyl™, Citrozym™, Denimax™, Dezyme™, Dextrozyme™,Duramyl™, Energex™, Finizym™, Fungamyl™, Gamanase™, Glucanex™,Lactozym™, Liquezyme™, Maltogenase™, Natalase™, Pentopan™, Pectinex™,Promozyme™, Pulpzyme™, Novamyl™, Termamyl™, AMG™ (AmyloglucosidaseNovo), Maltogenase™, Sweetzyme™ and Aquazym™ (all available fromNovozymes A/S). Further carbohydrases are available from othersuppliers, such as the Roxazyme™ and Ronozyme™ product series (DSMNutritional Products), the Avizyme™, Porzyme™ and Grindazyme™ productseries (Danisco, Finnfeeds), and Natugrain™ (BASF), Purastar™ andPurastar™ OxAm (Genencor).

Other commercially available enzymes include Mannaway™, Pectaway™,Stainzyme™ and Renozyme™.

In a particular embodiment of the present invention the feed enzyme isselected from the group consisting of endoglucanases,endo-1,3(4)-beta-glucanases, proteases, phytases, galactanases,mannanases, dextranases and alpha-galactosidase, and reference is madeto WO 2003/062409 which is hereby incorporated by reference.

Particular suitable feed enzymes include: amylases, phosphotases, suchas phytases, and/or acid phosphatases; carbohydrases, such as amylolyticenzymes and/or plant cell wall degrading enzymes including cellulasessuch as β-glucanases and/or hemicellulases such as xylanases orgalactanases; proteases or peptidases such as lysozyme; galactosidases,pectinases, esterases, lipases, in particular phospholipases such as themammalian pancreatic phospholipases A2 and glucose oxidase. Inparticular, the feed enzymes have a neutral and/or acidic pH optimum. Ina particular embodiment of the present invention the feed enzyme isselected from the group consisting of amylases, phosphotases, phytases,cellulases, β-glucanases, hemicellulases, proteases, peptidases,galactosidases, pectinases, esterases, lipases and glucose oxidase.

In a particular embodiment of the present invention the enzyme isselected from the group consisting of amylases, proteases,beta-glucanases, phytases, xylanases, phospholipases and glucoseoxidases.

Zinc Salts of Organic Acids

The zinc salt of an organic acid is in a particular embodiment watersoluble.

When working with feed for animals it is important that the materialsused in the granule have a certain purity thus in one embodiment of thepresent invention the zinc salt of organic acid is food grade. Theorganic zinc salt of an organic acid may be selected but is not limitedto the group consisting of zinc salts of citrate, malate, maleate,malonate, methionate, succinate, lactate, formate, acetate, butyrate,propionate, benzoate, tartrate, ascorbate, gluconate, zinc chelates ofamino acids hydrates and combinations thereof.

In a particular embodiment of the present invention the zinc salt of anorganic acid is selected from the group consisting of zinc salts ofcitrate, malate, maleate, malonate, methionate, succinate, lactate,formate, acetate and zinc chelates of amino acids hydrates. The zincsalt of an organic acid may be selected from the group consisting ofzinc citrate, zinc malate, zinc maleate, zinc malonate, zinc methionate,zinc succinate, zinc lactate, zinc formate, zinc acetate, zinc butyrate,zinc propionate, zinc benzoate, zinc tatrate, zinc ascorbate, zincgluconate, zinc methionate, zinc lysine, zinc methionine andcombinations thereof.

The zinc salt of an organic acid may be selected from the groupconsisting of zinc citrate, zinc malate, zinc maleate, zinc malonate,zinc methionate, zinc succinate, zinc lactate, zinc formate, zincacetate, zinc butyrate, zinc propionate, zinc benzoate, zinc tatrate,zinc ascorbate, zinc gluconate, zinc methionate, zinc lysine andcombinations thereof.

The zinc salt of an organic acid may be selected from the groupconsisting of zinc citrate zinc malate, zinc maleate, zinc malonate,zinc methionate, zinc succinate, zinc lactate, zinc formate, zincacetate, zinc butyrate, zinc propionate, zinc benzoate, zinc tatrate,zinc ascorbate, zinc gluconate, zinc methionate, zinc lysine, zincmethionine and combinations thereof.

In a particular embodiment of the present invention the organic anion isselected from carboxylates. In a particular embodiment, the zinc salt isselected from the group consisting of zinc citrate, zinc malate, zincmaleate, zinc malonate, zinc methionate, zinc succinate, zinc lactate,zinc formate, zinc acetate, zinc butyrate, zinc propionate, zincbenzoate, zinc tatrate, zinc ascorbate, zinc gluconate, zinc methionateand combinations thereof.

In a particular embodiment of the present invention the zinc salt of anorganic acid is selected from the group consisting of zinc salts ofsimple organic acids (less than 10 carbon atoms).

In a particular embodiment of the present invention the zinc salts oforganic acids includes zinc salts of aminoacids such as zinc lysinechelate or zinc methionine chelate. In a particular embodiment, the zincsalt of an organic acid is not including aminoacids.

In a more particular embodiment of the present invention the zinc saltof an organic acid is selected from the group consisting of zinccitrate, zinc acetate, zinc methionate, zinc methionine chelate andcombinations thereof.

The amount of zinc salt of an organic acid added is in a particularembodiment of the present invention 0.01-15% w/w based on the enzymecore. In a more particular embodiment of the present invention the zincsalt of an organic acid is added in amount of 0.05-10% w/w on the enzymecore. In an even more particular embodiment of the present invention thezinc salt of an organic acid is added in amount of 0.1-7% w/w. In a mostparticular embodiment of the present invention the zinc salt of anorganic acid is added in amount of 0.5-3.5% w/w of the enzyme core. Theamounts are calculated on water free zinc salts. By water free is meantnot including crystal water present in the salt.

In a particular embodiment of the present invention the zinc salt of anorganic acid may be selected from the group consisting of zinc citrate,zinc malate, zinc maleate, zinc malonate, zinc methionate, zincsuccinate, zinc lactate, zinc formate, zinc acetate, zinc butyrate, zincpropionate, zinc benzoate, zinc tatrate, zinc ascorbate, zinc gluconate,zinc methionate, zinc lysine and combinations thereof, and the zinccompound is added in an amount of 0.01-15% w/w based on the enzyme core.

The zinc salt of an organic acid may be added in the form of a solution,dispersion, emulsion or in solid form. In a particular embodiment of thepresent invention the zinc salt of an organic acid is added in form of asolution. The solution is in a particular embodiment an aqueoussolution. In a particular embodiment, the zinc salt of an organic acidis added as a liquid. In another embodiment, the zinc salt of an organicacid is added as a powder.

In a particular embodiment of the present invention the zinc salt of anorganic acid may be selected from the group consisting of zinc citrate,zinc malate, zinc maleate, zinc malonate, zinc methionate, zincsuccinate, zinc lactate, zinc formate, zinc acetate, zinc butyrate, zincpropionate, zinc benzoate, zinc tatrate, zinc ascorbate, zinc gluconate,zinc methionate, zinc lysine and combinations thereof, and the zinccompound is added in an amount of 0.01-15% w/w based on the enzyme coreand the zinc salt of an organic acid is added as a powder.

The zinc salt of an organic acid is in a particular embodiment presentin the granule together with the enzyme as a mixture. In anotherparticular embodiment of the present invention the zinc salt of anorganic acid is present in a layer surrounding the enzyme layer.

In a particular embodiment of the present invention the zinc salt of anorganic acid may be selected from the group consisting of zinc citrate,zinc malate, zinc maleate, zinc malonate, zinc methionate, zincsuccinate, zinc lactate, zinc formate, zinc acetate, zinc butyrate, zincpropionate, zinc benzoate, zinc tatrate, zinc ascorbate, zinc gluconate,zinc methionate, zinc lysine and combinations thereof, and the zinccompound is added in an amount of 0.01-15% w/w based on the enzyme coreand the zinc salt of an organic acid is present in granule together withthe enzyme as a mixture.

Additional Granulation Agents

The granule may comprise additional materials such as binders, fillers,fibre materials, stabilizing agents, solubilising agents, suspensionagents, viscosity regulating agents, light spheres, plasticizers, salts,lubricants and fragrances.

Binders of the present invention can be synthetic polymers, waxesincluding fats, fermentation broth, carbohydrates, salts orpolypeptides.

Synthetic Polymers

By synthetic polymers is meant polymers which backbone has beenpolymerized synthetically.

Suitable synthetic polymers of the invention include in particularpolyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), polyvinyl acetate,polyacrylate, polymethacrylate, poly-acrylamide, polysulfonate,polycarboxylate, and copolymers thereof, in particular water-solublepolymers or copolymers.

In a particular embodiment of the present invention the syntheticpolymer is a vinyl polymer.

Waxes

A “wax” in the context of the present invention is to be understood as apolymeric material having a melting point between 25-150° C.,particularly 30 to 100° C., more particularly 35 to 85° C., mostparticularly 40 to 75° C. The wax is preferably in a solid state at roomtemperature, 25° C. The lower limit is preferred to set a reasonabledistance between the temperature at which the wax starts to melt to thetemperature at which the granules or compositions comprising thegranules are usually stored, 20 to 30° C.

For some granules, a preferable feature of the wax is that the waxshould be water soluble or water dispersible, the wax shoulddisintegrate and/or dissolve providing a quick release and dissolutionof the active incorporated in the particles to the aqueous solution.Examples of water soluble waxes are poly ethylene glycols (PEG's).Amongst water insoluble waxes, which are dispersible in an aqueoussolution are triglycerides and oils. For some granules, it is preferablethat the wax is insoluble.

In a particular embodiment of the present invention the wax compositionis a hydrophilic composition. In a particular embodiment, at least 25%w/w of the constituents comprised in the wax composition is soluble inwater, preferably at least 50% w/w, preferably at least 75% w/w,preferably at least 85% w/w, preferably at least 95% w/w, preferably atleast 99% w/w.

In another embodiment, the wax composition is hydrophilic anddispersible in an aqueous solution.

In a particular embodiment, the wax composition comprises less than 75%w/w hydrophobic constituents, preferably less than 50% w/w, preferablyless than 25% w/w, preferably less than 15% w/w, preferably less than 5%w/w, preferably less than 1% w/w.

In a particular embodiment, the wax composition comprise less than 75%w/w water insoluble constituents, preferably less than 50% w/w,preferably less than 25% w/w, preferably less than 15% w/w, preferablyless than 5% w/w, preferably less than 1% w/w.

Suitable waxes are organic compounds or salts of organic compoundshaving one or more of the above-mentioned properties.

The wax composition of the invention may comprise any wax, which ischemically synthesized. It may also equally well comprise waxes isolatedfrom a natural source or a derivative thereof. Accordingly, the waxcomposition of the invention may comprise waxes selected from thefollowing non-limiting list of waxes.

-   -   Polyethylene glycols, PEG. Different PEG waxes are commercially        available having different molecular sizes, wherein PEG's with        low molecular sizes also have low melting points. Examples of        suitable PEG's are PEG 1500, PEG 2000, PEG 3000, PEG 4000, PEG        6000, PEG 8000, PEG 9000 etc., e.g., from BASF (Pluriol E        series) or from Clariant or from Ineos. Derivatives of Poly        ethylene glycols may also be used.    -   polypropylenes (e.g., polypropylene glycol Pluriol P series from        BASF) or polyethylenes or mixtures thereof.    -   Derivatives of polypropylenes and polyethylenes may also be        used.    -   Polymers of ethyleneoxide, propyleneoxide or copolymers thereof        are useful, such as in block polymers, e.g., Pluronic PE 6800        from BASF.    -   Derivatives of ethoxylated fatty alcohols.    -   Waxes isolated from a natural source, such as Carnauba wax        (melting point between 80-88° C.), Candelilla wax (melting point        between 68-70° C.) and bees wax. Other natural waxes or        derivatives thereof are waxes derived from animals or plants,        e.g., of marine origin. Hydrogenated plant oil or animal tallow.        Examples of such waxes are hydrogenated ox tallow, hydrogenated        palm oil, hydrogenated cotton seeds and/or hydrogenated soy bean        oil, wherein the term “hydrogenated” as used herein is to be        construed as saturation of unsaturated carbohydrate chains,        e.g., in triglycerides, wherein carbon=carbon double bonds are        converted to carbon-carbon single bonds. Hydrogenated palm oil        is commercially available, e.g., from Hobum Oele and Fette        GmbH—Germany or Deutche Cargill GmbH—Germany.    -   Fatty acid alcohols, such as the linear long chain fatty acid        alcohol NAFOL 1822 (C18, 20, 22) from Condea Chemie        GMBH—Germany, having a melting point between 55-60° C.        Derivatives of fatty acid alcohols.    -   Mono-glycerides and/or di-glycerides, such as glyceryl stearate,        wherein stearate is a mixture of stearic and palmitic acid, are        useful waxes. An example of this is Dimodan PM—from Danisco        Ingredients, Denmark.    -   Fatty acids, such as hydrogenated linear long chained fatty        acids and derivatives of fatty acids.    -   Paraffines, i.e., solid hydrocarbons.    -   Micro-crystalline wax.

In further embodiments waxes which are useful in the invention can befound in C. M. McTaggart et al., 1984, Int. J. Pharm. 19: 139 orFlanders et al., 1987, Drug Dev. Ind. Pharm. 13: 1001, both of which areincorporated herein by reference.

In a particular embodiment of the present invention the wax of thepresent invention is a mixture of two or more different waxes.

In a particular embodiment of the present invention the wax or waxes isselected from the group consisting of PEG, fatty acids, fatty acidalcohols and glycerides.

In another particular embodiment of the present invention the waxes arechosen from synthetic waxes. In a more particular embodiment the waxesof the present invention are PEG. In a most particular embodiment of thepresent invention the wax is selected from the group of beef tallow, PEGand palm oil.

Fermentation Broth

A fermentation broth in accordance with the invention comprisesmicrobial cells and/or cell debris thereof (biomass).

In a preferred embodiment, the fermentation broth comprises at least 10%of the biomass, more preferably at least 50%, even more preferably atleast 75% and most preferably at least 90% or at least 95% of thebiomass originating from the fermentation. In another preferredembodiment, the broth contains 0-31% w/w dry matter, preferably 0-20%w/w, more preferably 0-15% w/w such as 10-15% w/w dry matter, 0% drymatter being excluded from said ranges. The biomass may constitute up to90% w/w of the dry matter, preferably up to 75% w/w, more preferably upto 50% w/w of the dry matter, while the enzyme may constitute up to 50%w/w of the dry matter, preferably up to 25% w/w, more preferably up to10% w/w of the dry matter.

Polysaccharides

The polysaccharides of the present invention may be un-modifiednaturally occurring polysaccharides or modified naturally occurringpolysaccharides.

Suitable polysaccharides include cellulose, pectin, dextrin and starch.The starches may be soluble or insoluble in water.

In a particular embodiment of the present invention the polysaccharideis a starch. In a particular embodiment of the present invention thepolysaccharide is an insoluble starch.

Naturally occurring starches from a wide variety of plant sources aresuitable in the context of the invention (either as starches per se, oras the starting point for modified starches), and relevant starchesinclude starch from: rice, corn, wheat, potato, oat, cassava, sago-palm,yuca, barley, sweet potato, sorghum, yams, rye, millet, buckwheat,arrowroot, taro, tannia, and may for example be in the form of flour.

Cassava starch is among preferred starches in the context of theinvention; in this connection, it may be mentioned that cassava andcassava starch are known under various synonyms, including tapioca,manioc, mandioca and manihot.

As employed in the context of the present invention, the term “modifiedstarch” denotes a naturally occurring starch, which has undergone somekind of at least partial chemical modification, enzymatic modification,and/or physical or physicochemical modification, and which—ingeneral—exhibits altered properties relative to the “parent” starch.

In a particular embodiment of the present invention the granulecomprises a polysaccharide.

Salts

The core may comprise additional salt. The salt may be an inorganicsalt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate,chloride or carbonate or salts of simple organic acids (less than 10carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate oracetate. Examples of cations in these salts are alkali or earth alkalimetal ions, although the ammonium ion or metal ions of the firsttransition series, such as sodium, potassium, magnesium, calcium, zincor aluminium. Examples of anions include chloride, iodide, sulfate,sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasicphosphate, hypophosphite, dihydrogen pyrophosphate, carbonate,bicarbonate, metasilicate, citrate, malate, maleate, malonate,succinate, lactate, formate, acetate, butyrate, propionate, benzoate,tartrate, ascorbate or gluconate. In particular, alkali- or earth alkalimetal salts of sulfate, sulfite, phosphate, phosphonate, nitrate,chloride or carbonate or salts of simple organic acids such as citrate,malonate or acetate may be used. Specific examples include NaH₂PO₄,Na₂HPO₄, Na₃PO₄, (NH₄)H₂PO₄, K₂HPO₄, KH₂PO₄, Na₂SO₄, K₂SO₄, KHSO₄,ZnSO₄, MgSO₄, Mg(NO₃)₂, (NH₄)₂SO₄, sodium borate, magnesium acetate andsodium citrate.

The salt may also be a hydrated salt, i.e., a crystalline salt hydratewith bound water(s) of crystallization, such as described in WO99/32595. Examples of hydrated salts include magnesium sulfateheptahydrate (MgSO₄(7H₂O)), zinc sulfate heptahydrate (ZnSO₄(7H₂O)),sodium phosphate dibasic heptahydrate (Na₂HPO₄(7H₂O)), magnesium nitratehexahydrate (Mg(NO₃)₂(6H₂O)), sodium borate decahydrate, sodium citratedihydrate and magnesium acetate tetrahydrate.

In a particular embodiment of the present invention the binder is apolypeptide. The polypeptide may be selected from gelatin, collagen,casein, chitosan, poly aspartic acid and poly glutamatic acid. Inanother particular embodiment, the binder is a cellulose derivative suchas hydroxypropyl cellulose, methyl cellulose or CMC. A suitable binderis a carbohydrate binder such as dextrin e.g Glucidex 21D or Avedex W80.

Fillers

Suitable fillers are water soluble and/or insoluble inorganic salts suchas finely ground alkali sulphate, alkali carbonate and/or alkalichloride, clays such as kaolin (e.g., SPESWHITE™, English China Clay),bentonites, talcs, zeolites, chalk, calcium carbonate and/or silicates.

Typical fillers are di-sodium sulphate and calcium-lignosulphonate.Other fillers are silica, gypsum, kaolin, talc, magnesium aluminiumsilicate and cellulose fibers.

Fiber Materials

Pure or impure cellulose in fibrous form such as sawdust, pure fibrouscellulose, cotton, or other forms of pure or impure fibrous cellulose.Also, filter aids based on fibrous cellulose can be used. Several brandsof cellulose in fibrous form are on the market, e.g., CEPO™ andARBOCELL™. Pertinent examples of fibrous cellulose filter aids areARBOCELL BFC 200™ and ARBOCELL BC 200™. Synthetic fibres may also beused as described in EP 304331 B1.

Stabilizing Agents

Stabilizing or protective agents such as conventionally used in thefield of granulation. Stabilizing or protective agents may fall intoseveral categories: alkaline or neutral materials, reducing agents,antioxidants and/or salts of first transition series metal ions. Each ofthese may be used in conjunction with other protective agents of thesame or different categories. Examples of alkaline protective agents arealkali metal silicates, carbonates or bicarbonates. Examples of reducingprotective agents are salts of sulfite, thiosulfite, thiosulfate orMnSO₄ while examples of antioxidants are methionine, butylatedhydroxytoluene (BHT) or butylated hydroxyanisol (BHA). In particular,stabilizing agents may be salts of thiosulfates, e.g., sodiumthiosulfate or methionine. Still other examples of useful stabilizersare gelatine, urea, sorbitol, glycerol, casein, Poly vinyl pyrrolidone(PVP), hydroxypropylmethylcellulose (HPMC), carboxymethyl cellulose(CMC), hydroxyethylcellulose (HEC), powder of skimmed milk and/or edibleoils, such as soy oil or canola oil. Particular stabilizing agents infeed granules are a lactic acid source or starch. In a particularembodiment of the present invention the granule comprises a lactic acidsource according to patent application no. EP 1,117,771 which is herebyincorporated as reference. A preferred lactic acid source is corn steepliquor. It is also well known in the art that enzyme substrates such asstarch, lipids, proteins etc can act as stabilizers for enzymes.

Solubilizing Agents

As is known by the person skilled in the art, many agents, through avariety of methods, serve to increase the solubility of formulations,and typical agents known to the art can be found in NationalPharmacopeia's.

Light Spheres

Light spheres are small particles with low true density. Typically, theyare hollow spherical particles with air or gas inside. Such materialsare usually prepared by expanding a solid material. These light spheresmay be inorganic of nature or organic of nature. Polysaccharides arepreferred, such as starch or derivatives thereof. Biodac® is an exampleof non-hollow lightweight material made from cellulose (waste frompapermaking), available from GranTek Inc. These materials may beincluded in the granules of the invention either alone or as a mixtureof different light materials.

Suspension Agents

Suspension agents, mediators and/or solvents may be incorporated.

Viscosity Regulating Agents

Viscosity regulating agents may be present.

Plasticizers

Plasticizers of the present invention include, for example: polyols suchas sugars, sugar alcohols, glycerine, glycerol trimethylol propane,neopentyl glycol, triethanolamine, mono-, di- and triethylene glycol orpolyethylene glycols (PEGs) having a molecular weight less than 1000;urea and water.

Lubricants

As used in the present context, the term “lubricant” refers to anyagent, which reduces surface friction, lubricates the surface of thegranule, decreases tendency to build-up of static electricity, and/orreduces friability of the granules. Lubricants can serve asanti-agglomeration agents and wetting agents. Examples of suitablelubricants are lower polyethylene glycols (PEGs) and mineral oils. Thelubricant is particularly a mineral oil or a nonionic surfactant, andmore particularly the lubricant is not miscible with the othermaterials.

Coatings

The granules of the present invention may comprise one, two or moreadditional coating layers.

Coatings may be applied to the granule to provide additionalcharacteristics or properties. Thus, for example, an additional coatingmay achieve one or more of the following effects:

(i) reduction of the dust-formation tendency of a granule;

(ii) protection of the enzyme in the granule against hostile compoundsin the surroundings.

(iii) dissolution at a desired rate upon introduction of the granuleinto a liquid medium (such as an acid medium);

(iv) provide a better physical strength of the granule.

The coatings of the present invention generally are applied as one ormore layers surrounding the core. Embodiments include one, two, three orfour protective coating layers. Suitable coating materials are polymers,carbohydrates, proteins, lipids, fats and oils, fatty acids, inorganicsalts, and gums and mixtures thereof.

The coatings include moisture barrier coatings and moisture hydratingcoatings. The moisture barrier coatings function by excluding moisture,for instance by forming a shell layer that typically does not absorbmoisture and prevents or retards the rate of moisture migration into thegranule. Moisture hydrating coatings on the granule absorb or bindmoisture as either free water or water of hydration, thereby acting toimpede or retard the extent or rate of transport of external moistureinto the granule. The moisture hydrating coatings typically constituteat least about 35% w/w of the granule. The moisture hydrating materialsin the coatings thermally insulate the enzymes and will absorb a certainamount of moisture and retain it within the hydrating material withoutallowing it to pass through into the portion of the granule having theenzyme. For moisture hydrating coatings on stable, granules that do notcontain appreciable amounts of water prior to steam treatment, suchcoatings may constitute about 25% w/w of the granule. Moisture barriercoatings typically comprise hydrophobic materials, such as hydrophobicpolymers, for example PVA, HPMC, acid-thinned hydroxypropyl starches andoxidized starch; proteins, for example whey and whey proteinconcentrates; lipids, for example, lecithin; fats and oils, fatty acids,latex and gums, for example, gum arabic. Certain moisture barriercoatings, such as PVA and gum arabic, are not readily oxidized and findparticularly applicability in providing chemical stability when thegranules of the invention are stored in unpelleted or untabletedmixtures, for instance, in premixes that contain choline chloride.Moisture hydrating coating materials typically are hydrophilicmaterials, such as carbohydrates and inorganic salts, including hydratedsalts. Examples of moisture hydrating materials are magnesium sulfate,sodium sulfate, maltodextrin, ammonium sulfate, sugars, for example,sucrose, and native cornstarch. Polymers used for the protectivecoatings are polyvinyl alcohol (PVA), polyethylene glycol, polyvinylpyrrolidone, polyacrylates, polyethylene oxides (PEO), polylactic acid,polyvinylcloride, polyvinylacetate, polyvinyl pyrrolidones (PVP),cellulose ethers, alginates, gelatin, modified starches and substitutedderivatives, hydrolysates and copolymers thereof, such as acid-thinnedhydroxypropyl starch, such has, Pure Cote™ hydroxypropyl methylcellulose (HPMC), methyl cellulose (MC), carboxymethyl cellulose (CMC),and ethyl cellulose. Most preferred polymers for the protective coatingsare PVA, modified PVA, as described in U.S. Pat. No. 6,872,696, andmodified cellulose, such as methyl cellulose and hydroxylpropylmethylcellulose, as described in WO 99/51210, both of which are incorporatedby reference herein. Carbohydrates used for the protective coatings aremaltodextrin hydroxylmethyl cellulose, modified or native starches madefrom corn, sorghum, arrowroot, rice, wheat, rye, barley, oat, potato,yam, tapioca, cassava, sago, and sugars including sucrose, corn syrupsolids, molasses, glucose, fructose, and lactose. Proteins used for theprotective coatings are whey powder, whey protein concentrate, wheyprotein isolate, caseinates, soy protein concentrate and isolate, zein,albumin and gelatin.

Simple, compound and derived lipids that may be used in the protectivecoatings are waxes (for example, vegetable, mineral and synthetic, suchas carnauba, candelilla, beeswax, cerumen, carnuba, shellac, paraffin,and microcrystalline waxes); lecithin (for example mono- anddiglycerides); fatty acids (for example stearic, palmitic, linoleic,oleic, butyric, and arachidonic fatty acids and their salts of sodium,potassium, calcium and zinc); and fats and oils (for example,hydrogenated or partially hydrogenated fats and oils, such as soy, corn,cottonseed, tallow, canola, and linseed oil). A preferred lipid for theprotective coatings is lecithin.

Inorganic salts used for the protective coatings include salts ofsulfate, citrate, chloride, carbonate, sulfite, phosphate, phosphonate,and bicarbonate salts of sodium, ammonium, potassium, calcium, magnesiumand zinc. Preferred salts are magnesium, sodium and ammonium sulfates.

Gums that may be used in the protective coatings include gum arabic,guar gum, agar, gum tragacanth, karya gum, locust bean gum, carageenan,xanthan gum, and alginates.

The protective coatings of the present invention may further includeplasticizers, lubricants, pigments and powders, such as talc, bentonite,kaolin, corn starch, magnesium silicate, calcium carbonate, andchitosan.

Certain embodiments of the present invention typically have a singlelayer of a moisture hydrating material that is approximately at least55% w/w of the granule. Because the capacity of moisture hydratingcoatings to take up and sequester water has a limit, relatively highlevels of single layer coatings are applied. Alternatively, moisturehydrating material(s) may be applied in two layers. Other embodiments ofthe present invention have protective coatings utilizing both moisturehydrating materials and moisture barrier materials. In theseembodiments, the amount of moisture hydrating material may be lower, atleast about 25% w/w of the granule and the moisture barrier material isabout 2% to 25% w/w of the granule. Using both moisture hydratingmaterials and moisture barrier materials combines protective mechanismsand typically reduces cost, particularly of the moisture barriermaterials.

Moisture barrier materials, particularly film-forming materials may besubject to mechanical damage which, if these materials are used alone asa thin coating, may lead to loss of protection for the enzyme. Thecombination allows for the use of less of both materials than would berequired if the materials were used alone. The combination allows forsome damage to the moisture barrier layer in view of the presence of themoisture hydrating material.

Any conventional coating(s) of desired properties may be applied andexamples of conventional coating materials and coating methods is, interalia, described in U.S. Pat. No. 4,106,991, EP 170360, EP 304332, EP304331, EP 458849, EP 458845, WO 97/39116, WO 92/12645, WO 89/08695, WO89/08694, WO 87/07292, WO 91/06638, WO 92/13030, WO 93/07260, WO93/07263, WO 96/38527, WO 96/16151, WO 97/23606, U.S. Pat. Nos.5,324,649, 4,689,297, EP 206417, EP 193829, DE 4344215, DE 4322229 A, DD263790, JP 61162185, JP 58179492 or POT/DK/01/00628.

In a particular embodiment of the present invention the additionalcoating is a wax coating, according to U.S. Pat. No. 4,106,991 or EP0,569,468 which is hereby incorporated by reference. For suitable waxessee the section “Waxes” above. In a particular embodiment of the presentinvention an additional coating may comprise PVA, PEG, beef tallowand/or palm oil. Optionally, the granules can be coated with a coatingmixture.

In a particular embodiment of the present invention the granule iscoated with a salt coating. The salt may be selected from the section“salts”.

Additional Coating Materials

The coating may comprise additional coating materials such as binders,fillers, fibre materials, enzyme stabilizing agents, salts, solubilizingagents, suspension agents, viscosity regulating agents, light spheres,plasticizers, salts, lubricants and fragrances as mentioned in thesection “additional granulation agents” above. Further coatingingredients may be pigments.

Pigments

Suitable pigments include, but are not limited to, finely dividedwhiteners, such as titanium dioxide or kaolin, coloured pigments, watersoluble colorants, as well as combinations of one or more pigments andwater-soluble colorants.

Preparation of the Enzyme Core

The enzyme core comprises an enzyme and a zinc salt of an organic acid.

Methods for preparing the enzyme core may be found in Handbook of PowderTechnology; Particle size enlargement by C. E. Capes; Volume 1; 1980;Elsevier. Preparation methods include known feed and granule formulationtechnologies, i.e.:

a) Spray dried products, wherein a liquid enzyme-containing solution isatomized in a spray drying tower to form small droplets which duringtheir way down the drying tower dry to form an enzyme-containingparticulate material. Very small particles can be produced this way(Michael S. Showell (editor); Powdered detergents; Surfactant ScienceSeries; 1998; vol. 71; page 140-142; Marcel Dekker).

b) Layered products, wherein the enzyme is coated as a layer around apre-formed inert core particle, wherein an enzyme-containing solution isatomized, typically in a fluid bed apparatus wherein the pre-formed coreparticles are fluidized, and the enzyme-containing solution adheres tothe core particles and dries up to leave a layer of dry enzyme on thesurface of the core particle. Particles of a desired size can beobtained this way if a useful core particle of the desired size can befound. This type of product is described in, e.g., WO 97/23606.

c) Absorbed core particles, wherein rather than coating the enzyme as alayer around the core, the enzyme is absorbed onto and/or into thesurface of the core. Such a process is described in WO 97/39116.

d) Extrusion or pelletized products, wherein an enzyme-containing pasteis pressed to pellets or under pressure is extruded through a smallopening and cut into particles which are subsequently dried. Suchparticles usually have a considerable size because of the material inwhich the extrusion opening is made (usually a plate with bore holes)sets a limit on the allowable pressure drop over the extrusion opening.Also, very high extrusion pressures when using a small opening increaseheat generation in the enzyme paste, which is harmful to the enzyme.(Michael S. Showell (editor); Powdered detergents; Surfactant ScienceSeries; 1998; vol. 71; page 140-142; Marcel Dekker).

e) Prilled products, wherein an enzyme powder is suspended in molten waxand the suspension is sprayed, e.g., through a rotating disk atomiser,into a cooling chamber where the droplets quickly solidify (Michael S.Showell (editor); Powdered detergents; Surfactant Science Series; 1998;vol. 71; page 140-142; Marcel Dekker). The product obtained is onewherein the enzyme is uniformly distributed throughout an inert materialinstead of being concentrated on its surface. Also U.S. Pat. Nos.4,016,040 and 4,713,245 are documents relating to this technique.

f) Mixer granulation products, wherein an enzyme liquid is added to adry powder composition of conventional granulating components. Theliquid and the powder in a suitable proportion are mixed and as themoisture of the liquid is absorbed in the dry powder, the components ofthe dry powder will start to adhere and agglomerate and particles willbuild up, forming granulates comprising the enzyme. Such a process isdescribed in U.S. Pat. No. 4,106,991 (Novo Nordisk) and relateddocuments EP 170360 B1 (Novo Nordisk), EP 304332 B1 (Novo Nordisk), EP304331 (Novo Nordisk), WO 90/09440 (Novo Nordisk) and WO 90/09428 (NovoNordisk). In a particular product of this process wherein varioushigh-shear mixers can be used as granulators, granulates consisting ofenzyme, fillers and binders etc. are mixed with cellulose fibres toreinforce the particles to give the so-called T-granulate. Reinforcedparticles, being more robust, release less enzymatic dust.

g) Size reduction, wherein the cores are produced by milling or crushingof larger particles, pellets, tablets, briquettes etc. containing theenzyme. The wanted core particle fraction is obtained by sieving themilled or crushed product. Over and undersized particles can berecycled. Size reduction is described in (Martin Rhodes (editor);Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons).

h) Fluid bed granulation. Fluid bed granulation involves suspendingparticulates in an air stream and spraying a liquid onto the fluidizedparticles via nozzles. Particles hit by spray droplets get wetted andbecome tacky. The tacky particles collide with other particles andadhere to them and form a granule.

i) The cores may be subjected to drying, such as in a fluid bed drier.Other known methods for drying granules in the feed or enzyme industrycan be used by the skilled person. The drying preferably takes place ata product temperature of from 25 to 90° C. For some enzymes, it isimportant the cores comprising the enzyme contain a low amount of waterbefore coating with the salt. If water sensitive enzymes are coated witha salt before excessive water is removed, it will be trapped within thecore and it may affect the activity of the enzyme negatively. Afterdrying, the cores preferably contain 0.1-10% w/w water.

Coating of the Granules

Conventional coatings and methods as known to the art may suitably beused, such as the coatings described in Danish PA 2002 00473, WO89/08694, WO 89/08695, 270 608 B1 and/or WO 00/01793. Other examples ofconventional coating materials may be found in U.S. Pat. No. 4,106,991,EP 170360, EP 304332, EP 304331, EP 458849, EP 458845, WO 97/39116, WO92/12645, WO 89/08695, WO 89/08694, WO 87/07292, WO 91/06638, WO92/13030, WO 93/07260, WO 93/07263, WO 96/38527, WO 96/16151, WO97/23606, WO 01/25412, WO 02/20746, WO 02/28369, U.S. Pat. Nos.5,879,920, 5,324,649, 4,689,297, 6,348,442, EP 206417, EP 193829, DE4344215, DE 4322229 A, DE 263790, JP 61162185 A and/or JP 58179492.

The coating may be prepared by the same methods as mentioned above inthe section “Preparation of the enzyme core”.

The granules obtained can be subjected to rounding off (e.g.,spheronization), such as in a Marumeriser™, or compaction.

The granules can be dried, such as in a fluid bed drier. Other knownmethods for drying granules in the feed or enzyme industry can be usedby the skilled person. The drying preferably takes place at a producttemperature of from 25 to 90° C.

Manufacturing of Feed Pellets

In the manufacturing of feed pellets, it is preferred to involve steamtreatment prior to pelleting, a process called conditioning. In thesubsequent pelleting step, the feed is forced through a die and theresulting strands are cut into suitable pellets of variable length.During this conditioning step the process temperature may rise to60-100° C.

The feed mixture (mash feed) is prepared by mixing the granulescomprising the feed enzyme with desired feed components. The mixture isled to a conditioner, e.g., a cascade mixer with steam injection. Thefeed is in the conditioner heated up to a specified temperature, 60-100°C., e.g., 60° C., 70° C., 80° C., 90° C. or 100° C. by injecting steam,measured at the outlet of the conditioner. The residence time can bevariable from seconds to minutes and even hours. Such as 5 seconds, 10seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10minutes, 15 minutes, 30 minutes and 1 hour. In a particular embodimentof the present invention the temperature is 100° C. and the residencetime is 60 seconds.

In a particular embodiment of the present invention the processtemperature during steam treatment is at least 60° C. In a moreparticular embodiment of the present invention the process temperatureduring steam treatment is at least 70° C. In an even more particularembodiment of the present invention the process temperature during steamtreatment is at least 80° C. In a most particular embodiment of thepresent invention the process temperature during steam treatment is atleast 90° C.

From the conditioner, the feed is led to a press, e.g., a Simon Heesenpress, and pressed to pellets with variable length, e.g., 15 mm. Afterthe press, the pellets are placed in an air cooler and cooled for aspecified time, e.g., 15 minutes.

A particular embodiment of the present invention is a method formanufacturing a feed composition comprising the steps of:

i. mixing feed components with granules of the invention,

ii. steam treating said composition (i), and

iii. pelleting said composition (ii).

In a particular embodiment of the present invention the granule of thepresent invention is steam treated and/or pelleted.

Animal Feed

The granule of the present invention is suitable for use in animal feedcompositions. The granule is mixed with feed components, such feedcomposition is called mash feed. The characteristics of the granuleallows its use as a component of a composition which is well suited asan animal feed, which is steam treated and subsequently pelletized. In aparticular embodiment the invention is a steam treated pelletized feedcomposition comprising a granule comprising a feed enzyme and a zincsalt of an organic acid.

The term animal includes all animals. Examples of animals arenon-ruminants, and ruminants, such as cows, sheep and horses. In aparticular embodiment, the animal is a non-ruminant animal. Non-ruminantanimals include mono-gastric animals, e.g., pigs or swine (including,but not limited to, piglets, growing pigs, and sows); poultry such asturkeys and chicken (including but not limited to broiler chickens,layers); young calves; and fish (including but not limited to salmon).

The feed of the present invention may comprise vegetable proteins. Theterm vegetable proteins as used herein refers to any compound,composition, preparation or mixture that includes at least one proteinderived from or originating from a vegetable, including modifiedproteins and protein-derivatives. In particular embodiments, the proteincontent of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60%(w/w).

Vegetable proteins may be derived from vegetable protein sources, suchas legumes and cereals, for example materials from plants of thefamilies Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, andPoaceae, such as soy bean meal, lupin meal and rapeseed meal.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g., soybean, lupine,pea, or bean.

In another particular embodiment, the vegetable protein source ismaterial from one or more plants of the family Chenopodiaceae, e.g.,beet, sugar beet, spinach or quinoa.

Other examples of vegetable protein sources are rapeseed, and cabbage.

Soybean is a preferred vegetable protein source.

Other examples of vegetable protein sources are cereals such as barley,wheat, rye, oat, maize (corn), rice, and sorghum.

Suitable animal feed additives are enzyme inhibitors, fat-solublevitamins, water soluble vitamins, trace minerals and macro minerals.

Further, optional, feed-additive ingredients are colouring agents, aromacompounds, stabilisers, antimicrobial peptides, and/or at least oneother enzyme selected from amongst phytases EC 3.1.3.8 or 3.1.3.26;xylanases EC 3.2.1.8; galactanases EC 3.2.1.89; and/or beta-glucanasesEC 3.2.1.4.

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A,Tritrpticin, Protegrin-1, Thanatin, Defensin, Ovispirin such asNovispirin (Robert Lehrer, 2000), and variants, or fragments thereofwhich retain antimicrobial activity.

Examples of anti-fungal polypeptides (AFP's) are the Aspergillusgiganteus, and Aspergillus niger peptides, as well as variants andfragments thereof which retain antifungal activity, as disclosed in WO94/01459 and PCT/DK02/00289.

Usually fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed.

The following are non-exclusive lists of examples of these components:

Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E,and vitamin K, e.g., vitamin K3.

Examples of water-soluble vitamins are vitamin B12, biotin and choline,vitamin B1, vitamin B2, vitamin B6, niacin, folic acid andpanthothenate, e.g., Ca-D-panthothenate.

Examples of trace minerals are manganese, zinc, iron, copper, iodine,selenium, and cobalt.

Examples of macro minerals are calcium, phosphorus and sodium.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-10% fish meal; and/or 0-20% whey.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

Methods and Materials Steam Test

A laboratory scale steam treatment test has been developed to simulatethe residual activities that are found after steam pelleting in largescale.

The laboratory scale steam treatment test comprises a 5 liter Lödigemixer equipped with a jacket. The mixer also has an opening in thebottom, which may be used for a thermocouple or introduction of steamthrough a pipe. Steam flow for treating the granulates are controlled byletting the steam at 1.5 bar pass a 2.0 mm hole. The mixer is preheatedby steam through the mixer jacket to reach a temperature of 100° C. Toavoid condensate in steam-connections and pipe to be inserted into themixer, a steam flush is allowed prior to the introduction of thegranulate. The tests conditions comprise a series of actions preciselycontrolled time wise for good reproducibility. At time zero, 1 kg ofenzyme granulates is added to the mixer while rotating the mixing toolsat full speed. Immediately after the steam-pipe is inserted into themixer for the granulates to reach 100° C., and after precisely 30seconds steam is turned off. The mixture is maintained at 100° C. foranother 30 seconds after which approx 200 gram is poured on to a 75micron sieving net. To immediately cool the warm granulate a net ispositioned on top of the sample net and the sample is cooled during 60second by use of a lid connected to air ventilation. A reference sampleis taken prior and after steam treatment and send to FYT activitymeasurement for residual activity to be calculated.

Measurements of Pelleting Stability

Experimental Set-Up in Examples 5, 8 and 11:

Approximately 50 g enzyme granulate was pre-mixed with 10 kg feed for 10minutes in a small horizontal mixer. This premix was mixed with 90 kgfeed for 10 minutes in a larger horizontal mixer. From the mixer, thefeed was led to the conditioner (a cascade mixer with steam injection)at a rate of approximately 300 kg/hour. The conditioner heated up thefeed to 100° C. (measured at the outlet) by injecting steam. Theresidence time in the conditioner was 60-70 seconds. From theconditioner, the feed was led to a Simon Heesen press equipped with3.0×35 mm horizontal die and pressed to pellets with a length of around15 mm. After the press, the pellets were placed in an air cooler andcooled for 15 minutes.

Feed Formulation:

   74.0% Grind corn 20.7% Toasted soy grits  5.0% soy oil  0.3% SolivitMikro 106 premix of minerals and vitamins  12% water content

Phytase Activity Analysis

Method: Phytase splits phytic acid into phosphate, released phosphate isreacted with vanadium and molydenium oxides into a colored (yellow)complex. Absorbance is measured at 415 nm.

Unit: 1 FTU=amount of enzyme which at standard conditions (as givenbelow) releases phosphate equivalent to 1 μM phosphate per minute.

Buffers:

Extraction buffer: 0.01% Tween 20 (polyoxyethylene sorbitan monolaurate)

Substrate: 5 mM phytic acid, 0.22 M acetate (sodium acetate/aceticacid), pH 5.5.

Reagent: 5 mM ammonium vanadate, 20 mM ammonium heptamolybdatetetrahydrate, 40 mM ammonia, 2.4 M nitric acid

Procedure:

Extraction of feed: 50 g feed is extracted in 500 ml extraction bufferfor 1 hour. Eventual further dilution in extraction buffer if theactivity is higher than 2.5 FTU/g feed. (Detection level is 0.1 FTU/gfeed). The sample is centrifuged (15 minutes at 4000 rpm). 300 μlsupernatant is mixed with 3 ml substrate and reacted for 60 minutes at37° C. 2 ml reagent is added. Samples are centrifuged (10 minutes at4000 rpm.). Absorbance at 415 nm is measured. Activity is determinedrelative to a standard curve prepared with KH₂PO₄.

Reference is made to WO 2003/66847.

EXAMPLES Example 1

A powder consisting of:

1.5 kg fibrous cellulose, Arbocel BC200

0.75 kg carbohydrate binder, Avedex W80

11.362 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.75 kg carbohydrate binder, Avedex W80

0.3 kg wheat starch

1.7 kg phytase concentrate

1.275 kg water

The granulation is performed in a manner as described in U.S. Pat. No.4,106,991, Example 1.

The obtained granulate is dried in a fluid bed to a water content below1% and sifted to obtain a product with the particle range 250 μm to 850μm. Finally, the product is coated with 11.5% palm oil and 22% calciumcarbonate in a manner as described in U.S. Pat. No. 4,106,991, Example22.

Example 2

A powder consisting of:

1.5 kg fibrous cellulose, Arbocel BC200

0.75 kg carbohydrate binder, Avedex W80

11.287 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.75 kg carbohydrate binder, Avedex W80

0.3 kg wheat starch

0.075 kg Zn-acetate×2 H₂O

1.7 kg phytase concentrate

0.215 kg 10% H₂SO₄

0.975 kg water

The granulation is performed in a manner as described in U.S. Pat. No.4,106,991, Example 1.

The granulate is dried in a fluid bed to a water content below 1% andsifted to obtain a product with the particle range 250 μm to 850 μm.Finally, the product is coated with 11% palm oil and 22% calciumcarbonate in a manner as described in U.S. Pat. No. 4,106,991, Example22.

Example 3

A powder consisting of:

1.5 kg fibrous cellulose, Arbocel BC200

0.75 kg carbohydrate binder, Avedex W80

11.122 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.75 kg carbohydrate binder, Avedex W80

0.3 kg wheat starch

0.240 kg Zn-acetate×2 H₂O

1.7 kg phytase concentrate

0.419 kg 10% H₂SO₄

0.75 kg water

The granulation is performed in a manner as described in U.S. Pat. No.4,106,991, Example 1.

The granulate is dried in a fluid bed to a water content below 1% andsifted to obtain a product with the particle range 250 μm to 850 μm.Finally, the product is coated with 11.0% palm oil and 22% calciumcarbonate in a manner as described in U.S. Pat. No. 4,106,991, Example22.

Example 4

A powder consisting of:

1.5 kg fibrous cellulose, Arbocel BC200

0.75 kg carbohydrate binder, Avedex W80

10.957 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.75 kg carbohydrate binder, Avedex W80

0.3 kg wheat starch

0.405 kg Zn-acetate×2 H₂O

1.7 kg phytase concentrate

0.438 kg 2 M H₂SO₄

0.75 kg water

The granulation is performed in a manner as described in U.S. Pat. No.4,106,991, Example 1.

The granulate is dried in a fluid bed to a water content below 1% andsifted to obtain a product with the particle range 250 μm to 850 μm.Finally, the product is coated with 11% palm oil and 22% calciumcarbonate in a manner as described in U.S. Pat. No. 4,106,991, Example22.

Example 5

The samples produced in Example 1 to Example 4 were tested in apelleting trial at 100° C., outlet of the conditioner. The phytasecontent was measured using analytical method EB-SM 0559.02 version 01(available from Novozymes upon request) prior to pelletizing and in thefeed pellets after pelletizing. The following residual activities of thephytase were found:

Residual activity of the Phytase in Batch [%] Example 1 68.5 Example 284.5 Example 3 84.1 Example 4 84.8

The conclusion is that Zn-acetate×2 H₂O significantly improves thepelleting stability compared to the reference Example 1 without addedzinc salt of an organic acid.

Example 6

A powder consisting of:

1.5 kg fibrous cellulose, Arbocel BC200

0.75 kg carbohydrate binder, Avedex W80

11.267 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.45 kg carbohydrate binder, Avedex W80

0.3 kg wheat starch

0.375 kg Zn-citrate×2 H₂O

1.8 kg phytase concentrate

0.033 kg 1 N H₂SO₄

1.025 kg water

The granulation is performed in a manner as described in U.S. Pat. No.4,106,991, Example 1.

The granulate was dried in a fluid bed to a water content below 1% andsifted to obtain a product with the particle range 250 μm to 850 μm.Finally, the product was coated with 10% palm oil and 14% kaolin in amanner as described in U.S. Pat. No. 4,106,991, Example 22.

Example 7

A powder consisting of:

1.5 kg fibrous cellulose, Arbocel BC200

0.75 kg carbohydrate binder, Avedex W80

11.342 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.6 kg carbohydrate binder, Avedex W80

0.3 kg wheat starch

0.150 kg Zn-citrate×2 H₂O

1.8 kg phytase concentrate

0.027 kg 1 N H₂SO₄

1.025 kg water

The granulation was performed in a manner as described in U.S. Pat. No.4,106,991, Example 1.

The granulate was dried in a fluid bed to a water content below 1% andsifted to obtain a product with the particle range 250 μm to 850 μm.Finally, the product was coated with 10.0% palm oil and 14% calciumcarbonate in a manner as described in U.S. Pat. No. 4,106,991, Example22.

Example 8

The samples produced in Example 6 and Example 7 were tested in apelleting trial at 100° C., outlet of the conditioner. The phytasecontent was measured using analytical method EB-SM 0559.02 version 01(available from Novozymes upon request) prior to pelletizing and in thefeed pellets after pelletizing. The following residual activities of thephytase were found:

Residual activity of the Phytase in Batch [%] Example 1 68.5 Example 682.4 Example 7 84.5

The conclusion is that Zn-citrate×2 H₂O significantly improves thepelleting stability compared to the reference Example 1.

Example 9

A powder consisting of:

1.5 kg fibrous cellulose, Arbocel BC200

0.75 kg carbohydrate binder, Avedex W80

11.212 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.75 kg carbohydrate binder, Avedex W80

0.3 kg wheat starch

0.150 kg Zn methionine chelate

1.7 kg phytase concentrate

1.125 kg water

The granulation was performed in a manner as described in U.S. Pat. No.4,106,991, Example 1.

The granulate was dried in a fluid bed to a water content below 1% andsifted to obtain a product with the particle range 250 μm to 850 μm.Finally, the product was coated with 11% palm oil and 22% calciumcarbonate in a manner as described in U.S. Pat. No. 4,106,991, Example22.

Example 10

A powder consisting of:

1.5 kg fibrous cellulose, Arbocel BC200

0.75 kg carbohydrate binder, Avedex W80

11.062 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.75 kg carbohydrate binder, Avedex W80

0.3 kg wheat starch

0.3 kg Zn methionine chelate

1.7 kg phytase concentrate

1.2 kg water

The granulation is performed in a manner as described in U.S. Pat. No.4,106,991, Example 1.

The granulate was dried in a fluid bed to a water content below 1% andsifted to obtain a product with the particle range 250 μm to 850 μm.Finally, the product was coated with 11.0% palm oil and 22% calciumcarbonate in a manner as described in U.S. Pat. No. 4,106,991, Example22.

Example 11

The samples produced in Example 9 and Example 10 were tested in apelleting trial at 100° C., outlet of the conditioner. The phytasecontent was measured using analytical method EB-SM 0559.02 version 01(available from Novozymes upon request) prior to pelletizing and in thefeed pellets after pelletizing. The following residual activities of thephytase were found:

Residual activity of the Phytase in Batch [%] Example 1 68.5 Example 985.4 Example 10 77.2

The conclusion is that Zn methionine chelate significantly improves thepelleting stability compared to the reference, Example 1.

Example 12

The per se stability was tested. Residual activity of the phytase in[%]:

After 4 weeks After 4 weeks After 4 weeks Batch at 40° C. at 50° C. at40° C./60% RH Example 1 88.5 81.9 51.9 Example 2 94.1 84.7 64.6 Example3 96.5 90.0 63.0 Example 4 97.7 88.6 52.6 Example 6 94.5 83.5 77.5Example 7 92.0 79.0 77.2 Example 9 95.9 84.0 80.4 Example 10 96.3 86.881.3

It is shown that especially zinc citrate and zinc methionine have verygood per se stability.

Example 13

Two granules were prepared: a granule comprising zinc acetate and agranule comprising zinc sulphate.

Granule 1:

A powder consisting of:

1.5 kg fibrous cellulose, Arbocel BC200

0.75 kg carbohydrate binder, Avedex W80

11.042 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.75 kg carbohydrate binder, Avedex W80

0.3 kg wheat starch

0.36 kg Zinc acetate×2H₂O

1.475 kg Phytase concentrate

1.10 kg water

Granule 2:

A powder consisting of:

1.5 kg fibrous cellulose, Arbocel BC200

0.75 kg carbohydrate binder, Avedex W80

10.868 kg finely ground sodium sulphate

was granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.75 kg carbohydrate binder, Avedex W80

0.3 kg wheat starch

0.534 kg Zinc sulphate×7H₂O

1.475 kg Phytase concentrate

1.20 kg water

The granulation was performed in a manner as described in U.S. Pat. No.4,106,991, Example 1.

The obtained granulate was dried in a fluid bed to a water content below1% and sifted to obtain a product with the particle range 250 μm to 700μm.

Residual activity after granulation and drying of the samples producedcompared to the dosed amount of Phytase Units were calculated. ThePhytase content was measured using analytical method EB-SM 0511(available from Novozymes upon request). The following residual activityof the Phytase were found:

Residual activity Batch [%] Granule 1 99 Granule 2 93

The conclusion is that Zn-acetate×2H₂O is supplying better stabilizationof the Phytase during granulation than Zn-sulphate×7H₂O.

Example 14

Two granules where prepared: a granule comprising zinc acetate and agranule comprising zinc sulphate.

Granule 1:

A powder consisting of:

1.5 kg fibrous cellulose, Arbocel BC200

0.75 kg carbohydrate binder, Avedex W80

10.592 kg finely ground sodium sulphate

was granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.75 kg carbohydrate binder, Avedex W80

0.3 kg wheat starch

0.45 kg ‘Corn Steep Liquor’ Powder

0.36 kg Zinc acetate×2H₂O

1.475 kg Phytase concentrate

0.850 kg water

Granule 2:

A powder consisting of:

1.5 kg fibrous cellulose, Arbocel BC200

0.75 kg carbohydrate binder, Avedex W80

10.418 kg finely ground sodium sulphate

was granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.75 kg carbohydrate binder, Avedex W80

0.3 kg wheat starch

0.45 kg ‘Corn Steep Liquor’ Powder

0.534 kg Zinc sulphate×7H₂O

1.475 kg Phytase concentrate

1.20 kg water

The granulation was performed in a manner as described in U.S. Pat. No.4,106,991, Example 1.

The obtained granules were dried in a fluid bed to a water content below1% and sifted to obtain a product with the particle range 250 μm to 700μm.

Residual activity after granulation and drying of the samples producedcompared to the dosed amount of Phytase Units were calculated. ThePhytase content was measured using analytical method EB-SM 0511(available from Novozymes upon request). The following residual activityof the Phytase were found:

Residual activity after granulation Batch [%] Granule 1 97 Granule 2 90

The conclusion is that Zn-acetate×2H₂O is supplying better stabilizationof the Phytase during granulation than Zn-sulphate×7H₂O.

Granules 1 and 2 were coated with 9-10% palm oil and 22% calciumcarbonate in a manner as described in U.S. Pat. No. 4,106,991, Example22.

The coated granules were then tested in a laboratory steam test exposingthe granulates for 30 seconds steam treatment and continued for another30 seconds at 100° C. before immediate air cooling. These conditionsresemble large scale pelleting conditions. The phytase content wasmeasured using analytical method EB-SM 0511 (available from Novozymesupon request). The following residual activities of the phytase werefound:

Residual activity of the Phytase in Batch [%] Granule 1 89 Granule 2 84

The conclusion was that Zn-acetate×2H₂O shows better stabilization ofthe enzyme during high temperature and humidity compared toZn-sulphate×7H₂O.

1-34. (canceled)
 35. A granule suitable for feed comprising a core and alayer surrounding the core, wherein the core comprises an enzyme and azinc salt of an organic acid, wherein the amount of zinc salt of anorganic acid is between 0.01-15% w/w based on the core.
 36. The granuleof claim 35, wherein the granule is steam treated.
 37. The granule ofclaim 35, wherein the amount of zinc salt of an organic acid added isbetween 0.05-10% w/w based on the enzyme core.
 38. The granule of claim35, wherein the amount of zinc salt of an organic acid added is between0.1-7% w/w based on the enzyme core.
 39. The granule of claim 35,wherein the amount of zinc salt of an organic acid added is between0.5-3.5% w/w based on the enzyme core.
 40. The granule of claim 35,wherein the zinc salt of an organic acid is selected from the groupconsisting of zinc salts of citrate, malate, maleate, malonate,methionate, succinate, lactate, formate, acetate, butyrate, propionate,benzoate, tartrate, ascorbate, gluconate, chelates of zinc andcombinations thereof.
 41. The granule of claim 40, wherein the chelatesof zinc is selected from the group consisting of zinc methioninechelate, zinc lysine chelate and combinations thereof.
 42. The granuleof claim 40, wherein the zinc salt of an organic acid is selected fromthe group consisting of zinc acetate, zinc citrate and zinc methionine.43. The granule of claim 35, wherein the enzyme is selected from thegroup consisting of amylases, cellulases, esterases, galactosidases,β-glucanases, glucose oxidases, hemicellulases, lipases, pectinases,peptidases, phosphotases, phytases, proteases, and mixtures thereof. 44.The granule of claim 35, wherein the enzyme is food grade.
 45. Thegranule of claim 35, wherein the zinc salt of an organic acid is foodgrade.
 46. The granule of claim 35, wherein the granule has a particlesize below 700 μm.
 47. The granule of claim 35, wherein the size of thegranule is between 210 and 390 μm.
 48. The granule of claim 35, whereinthe enzyme is thermo labile.
 49. The granule of claim 35, wherein thelayer comprises a salt coating.
 50. The granule of claim 35, wherein thelayer comprises a polymer coating.
 51. The granule of claim 35, whereinthe layer comprises a wax coating.
 52. The granule of claim 35, whereinthe granule further comprises a lactic acid source.
 53. A process ofpreparing the granule of claim 35, comprising the steps of: (a)preparing a core comprising a zinc salt of an organic acid and anenzyme; and (b) coating the core with a coating material.
 54. Theprocess of claim 53, wherein the granule is prepared in a mixer, a fluidbed, a fluidized spray dryer, a spray fluidizer, a spray dryer or anextruder.
 55. The process of claim 53, wherein the zinc salt of anorganic acid is a liquid.
 56. The process of claim 53, wherein the zincsalt of an organic acid is a powder.
 57. The process of claim 53,wherein the core comprises an inert particle.
 58. A feed compositioncomprising feed components and the granule of claim
 35. 59. The feedcomposition of claim 58, wherein the feed components are selected fromthe group consisting of vegetable proteins, fat-soluble vitamins, watersoluble vitamins, trace minerals, macro minerals and combinationsthereof.
 60. A pelletized feed composition comprising the granule ofclaim
 35. 61. A steam treated feed composition comprising the granule ofclaim
 35. 62. A method for feeding an animal, comprising administeringthe feed composition of claim 59 to the animal.
 63. A method formanufacturing a feed composition comprising the steps of: (a) mixingfeed components with the granule of claim 35 to form a mixture, (b)steam treating the mixture to form a steam-treated mixture, and (c)pelleting the steam-treated mixture.